Compounds modulators of VEGF activity and uses thereof

ABSTRACT

Compounds of general formula (1): X1Y1X2Y2X3Y3X4Y4Y5X5X6Y6X7Y7X8X9X10 wherein X1-X10 are any natural or unnatural amino acids and Y1 is Gln; Y2 is Met or Leu; Y3 is He; Y4 is Pro or Ser; Y5 is His or Gly; Y6 is Gln or Pro; Y7 is He or Tyr or their homolog or ortolog are described; these compounds are able to bind to the VEGF receptors and to modulate the angiogenesis mediated by the VEG.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to International Application NumberPCT/EP2010/060722 filed on Jul. 23, 2010 and U.S. ProvisionalApplication No. 61/228,384 filed on Jul. 24, 2009, the entire contentsof each of which are hereby incorporated by reference herein, in theirentirety and for all purposes.

FIELD OF THE INVENTION

The present invention relates to compounds which interact with VascularEndothelial Growth Factor (VEGF) receptors, modulate the VEGF dependentbiological response and their use as pharmacological agents to treatangiogenesis-dependent diseases. These compounds mimic the β-hairpinregion 79-92 of VEGF (or the corresponding fragment of Placenta GrowthFactor) which is involved in receptors recognition. Here are describedthe design and biological properties of these compounds and their usefor the treatments of pathologies dependent on the modulation of theVEGF biological activity, the diagnosis of pathologies which present aoverexpression of VEGF receptors and as biochemical tools for the studyof cellular pathway dependent on the activation of VEGF receptors.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 10541PTWO_seq listing_ST25.txt, created on Jul. 22,2010, with a size of 4,000 bytes. The Sequence Listing is incorporatedby reference herein.

STATE OF THE ART

Angiogenesis is a physiological process which refers to the remodelingof the vascular tissue characterized by the branching out of a new bloodvessel from a pre-existing vessel. It is intimately associated withendothelial cell (EC) migration and proliferation. ECs are particularlyactive during embryonic development while during adult life EC turnoveris very low and limited to particular physiological phenomena(Carmeliet, P. Nat Med 2003, 9, 653). In a healthy individualangiogenesis is finely tuned by pro- and anti-angiogenic factors, theshift from this equilibrium (angiogenic switch), under specific stimulisuch as hypoxia, is related to several human diseases (pathologicalangiogenesis) (Hanahan, D., Folkman, J. Cell 1996, 86, 353). Theprevalence of pro-angiogenic factors (excessive angiogenesis), isassociate with cancer, proliferating retinopathy, rheumatoid arthritisand psoriasis. Whereas, insufficient angiogenesis is at the basis ofcoronary diseases, ischemia and a reduced capacity for tissueregeneration (Carmeliet, P., Jain, R. K. Nature 2000, 407, 249).

One of the most studied angiogenic factor is the Vascular EndothelialGrowth Factor (VEGF). The VEGF and its receptors are overexpressed inpathological angiogenesis, making this system a target for therapeuticand diagnostic applications (Hanahan, D. et al. 1996 Cell 86, 353-364;Ferrara, N. et al. 1997 Endocr Rev 18, 4-25).

Vascular Endothelial Growth Factor

VEGF is a homodimeric glycoprotein, covalently bound by two disulfidebonds, characterized by a cystine knot motif. The vegf gene codify atleast for eight splicing isoforms differing in the number of amino acidsof the encoded polypeptide. The most common isoforms VEGF₁₂₁, VEGF₁₆₅,VEGF₁₈₉ and VEGF₂₀₆ are characterized by a different ability to bind toheparin, cell and matrices [148]. VEGF₁₂₁ is soluble and lack of theexons codifying for the heparin binding site; VEGF₁₆₅ is the mostabundant form in the majority of tissues and in pathologicalangiogenesis, it binds to heparin and is found partially bound to thematrix; VEGF₁₈₉ and VEGF₂₀₆ strongly interact with matrix and areactivated by proteolytic cleavage (Park et al., Mol Biol Cell 1993; 4:1317-26; Roth et al. Am J Pathol 2006; 68: 670-84). An additional splicevariant, VEGF_(165b), has been identified and resulted an endogenousinhibitor of VEGF (Woolard et al., Cancer Res 2004; 64: 7822-35).

VEGF possess several homologs: VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF andVEGF-E. They recognize different receptor and elicit diverse biologicalactivity. VEGF-A (or VEGF) is the main form, it is a mitogen specificfor vascular ECs and the main regulator of physiological andpathological angiogenesis. VEGF-B can form heterodimers with VEGF, itinduces expression of enzymes involved in ECM degradation and ECmigration, and it has a role in coronary artery development. VEGF-C and-D are mainly implicated in lymphagiogenesis; VEGF-C is mainly expressedduring embryogenesis whereas VEGF-D is also expressed in adult heart,lung and skeletal muscle (Carmeliet P. Nat Med 2003; 9:653-60). VEGF-Eis encoded by the parapoxvirus Orf virus. PlGF (Maglione et al., ProcNatl Acad Sci USA 1991; 88: 9267-71) is gaining much attention in thelast years because of its role in pathological angiogenesis (Luttun etal., Ann N Y Acad Sci 2002; 979: 80-93; Fischer et al., Cell 2007; 131:463-75; Luttun et al., Nat Med 2002; 8: 831-40). PlGF has beenimplicated in several physiological and pathological processes such asangiogenesis, arteriogenesis and atherosclerosis (Roy et al., FEBS Lett2006; 580: 2879-87). It is mainly expressed in placenta and itsexpression is upregulated during pathological conditions such asischemia and myocardial infarction (Iyer et al., Trends Cardiovasc Med2002; 12: 128-34). PlGF loss does not affect embryonic development butimpair angiogenesis during ischemia, wound healing, inflammation andcancer (Carmeliet et al., Nat Med 2001; 7: 575-83). Two main isoforms,PlGF-1 and PlGF-2, differing in the presence of high basic amino acidssequence and in the ability to bind to heparin have been described(Hauser S, Weich H A. Growth Factors 1993; 9: 259-68; Maglione et al.,Oncogene 1993; 8: 925-31).

The biological activity of the VEGF family is mediated through thebinding to three membrane receptors belonging to the tyrosine kinasefamily (VEGFR1, VEGFR2 and VEGFR3) and two receptors belonging to thesemaphorin family (NRP1 and NRP2). The function of each receptor inphysiological and pathological angiogenesis is still far to becompletely revealed. A detailed description of the biology andpathologies associated to VEGF and its receptors can be found elsewhere(Nagy et al., Annu Rev Pathol 2007; 2: 251-75; Roy et al., FEBS Lett2006; 580: 2879-87; Ferrara et al., Nat Med 2003; 9: 669-76; Ferrara N.Curr Opin Biotechnol 2000; 11: 617-24; Ferrara N, Davis-Smyth T. EndocrRev 1997; 18: 4-25; Cross et al., Trends Biochem Sci 2003; 28: 488-94;Veikkola et al., Cancer Res 2000; 60: 203-12; Hicklin D J, Ellis L M. JClin Oncol 2005; 23: 1011-27). VEGFR2 and VEGFR1 are mainly expressed onvascular endothelium but they have been found also on the surface ofnon-endothelial (Matsumoto T, Claesson-Welsh L. Sci STKE 2001; 2001:RE21) and tumor cells. Increased expression of these receptors occurs inresponse to several stimuli and results in priming of EC towardsproliferation, migration and angiogenesis (Ferrara, N. et al. 2003 NatMed 9, 669-676). They are composed of an intracellular kinase domain, atransmembrane region and an extracellular portion constituted of sevenimmunoglobulin-like domain. VEGF binds very tightly to both receptorsbut the binding affinity for VEGFR1 is ten times higher that for VEGFR2.They also differ in the ability to recognize VEGF homologs, in fact,VEGFR1 interacts also with PlGF and VEGF-B, whereas VEGFR2 recognizesVEGF-C, VEGF-D and VEGF-E. In a concise view of the receptors biology,the binding of VEGF induces receptor dimerization, autophosphorylationof the kinase domain and activation of several intracellular pathwaysending with mitogenesis, angiogenesis, permeability, survival andmigration. At embryonic stage VEGFR2 is essential for vasculogenesis andendothelial cells proliferations, while VEGFR1 is necessary for theorganization of the vascular system of the embryo (Ferrara et al., NatMed 2003; 9: 669-76). In the adult life VEGFR2 has been recognized asthe mayor mediator of VEGF biological activity on ECs whereas VEGFR1 hasbeen considered as a decoy receptor negatively regulating VEGF effects.Then, it has been showed that VEGFR1 is implicated in monocytesmigration (Hiratsuka et al., Proc Natl Acad Sci USA 1998; 95: 9349-54),hematopoiesis (Hattori et al., Nat Med 2002; 8: 841-9) and in releasingtissue specific growth-factors (Ferrara et al., Nat Med 2003; 9:669-76). Moreover, a cross-talk between the VEGF receptors, regulated byPlGF, was reported (Autiero et al. Nat Med 2003; 9: 936-43). Analternative splicing of VEGFR1 gene codifies for a receptor soluble formcontaining only six extracellular domain (sVEGFR1). This molecule bindsto VEGF, suppresses VEGF mitogenic activity and acts as an efficientspecific antagonist of VEGF in vivo (Kendall R L, Thomas K A. Proc NatlAcad Sci USA 1993; 90: 10705-9) Recently, it has been showed thatsVEGFR1 has a role in pre-eclampsia (Maynard et al., J Clin Invest 2003;111: 649-58).

VEGFR3 is found on the surface of lymphatic endothelial cells. Itselectively binds to VEGF-C and VEGF-D and mediates lymphagiogenesis(Oliver G, Alitalo K. Annu Rev Cell Dev Biol 2005; 21: 457-83; Alitaloet al., Nature 2005; 438: 946-53). VEGFR3 is believed to play diverseroles assisting cardiovascular development, remodeling of primaryvascular network during embryogenesis and facilitating lymphagiogenesisin adulthood [166]. Its activation has also been observed in someneoplastic conditions (Achen et al., J Pathol 2001; 193: 147-54; Valtolaet al., Am J Pathol 1999; 154: 1381-90).

NRP1 and NRP2, mainly implicated in axonal guidance and neuronaldevelopment, are also involved in physiological and pathologicalangiogenesis, in fact, they act as co-receptor for VEGF. They do nottransfer directly the intracellular signaling but enhance the bindingaffinity of the VEGF family members to VEGFR1 or VEGFR2. NRP1 binds toVEGF₁₆₅ and PlGF-2 and it is essential for neuronal and cardiovasculardevelopment, whereas NRP2 interact with VEGF₁₆₅ and VEGF-C and has arole in lymphagiogenesis (Pellet-Many et al., Biochem J 2008; 411:211-26; Geretti et al., Angiogenesis 2008; 11: 31-9).

Structural Data of VEGF Family

Many structural data of VEGF family members have been reported. SeveralVEGF or PlGF structures have been described either free (Muller, Y A etal., Structure 1997, 5, 1325; Muller, Y. A. et al., Proc Natl Acad SciUSA 1997, 94, 7192), bound to peptide inhibitors (Wiesmann, C. et al.,Biochemistry 1998, 37, 17765; Pan, B. et al., J Mol Biol 2002, 316, 769)or to a neutralizing antibody (Muller, Y A. et al., Structure 1998, 6,1153). The only structural data about VEGFR1 regards the domain 2 of theextracellular region, VEGFR1_(D2), in the free form (Starovasnik et al.,J Mol Biol 1999; 293: 531-44), bound to VEGF (Wiesmann, C. et al., Cell1997, 91, 695) or PlGF (Christinger et al., J Biol Chem 2004; 279:10382-8). So far, no structural data have been reported for VEGFR3 andthe extracellular domain of VEGFR2. Instead, VEGFR2 intracellular kinasedomain has been described (McTigue et al., Structure 1999; 7: 319-30). Apartial characterization of NRP1 and NRP2 receptors has been performed(Pellet-Many et al., Biochem J 2008; 411: 211-26). Very recently thecomplex between VEGF and the extracellular VEGFR1 domain has beenobserved by electron microscopy (Ruch et al., Nat Struct Mol Biol 2007;14: 249-50). VEGF is an antiparallel homodimer, covalently linkedthrough two disulfide bonds. It is characterized by a cystine knotmotif. The knot consists of two disulfide bridges, with a thirddisulfide bond passing through them. The structure of the ligands and ofthe receptor does not change moving from the free forms to the complexedones. Domain deletion studies on VEGFR1 and VEGFR2 showed that theligand binding site resides within the first three extracellular domainsand VEGFR1_(D2) binds VEGF about 60-times less tight than wild typeprotein, but its removal completely abolishes the binding. The overallstructure of the VEGF/VEGFR1_(D2) complex possesses approximately atwo-fold symmetry. The VEGF recognition interface is divided about 65%and 35% between both monomers. The analysis of structural andmutagenesis data allowed to identify residues involved in the binding tothe receptors. They are distributed over a discontinuous surface whichinclude residues from the N-terminal helix (17-25), the loop connectingstrand 133 to 134 (61-66) and strand 137 (103-106) of one monomer, aswell as residues from strand 132 (46-48) and from strand 135 and 136together with the connecting turn (79-91) of the other monomer. Therecognition interface is manly hydrophobic, except for the polarinteraction between Arg224 (VEGFR1) and Asp63 (VEGF). VEGFR2 and VEGFR1share the same VEGF binding region, in fact 5 out of 7 most importantVEGF binding residues are present in both interfaces (Muller, Y. A. etal., Proc Natl Acad Sci USA 1997, 94, 7192; Wiesmann, C. et al., Cell1997, 91, 695; Keyt et al., J Biol Chem 1996; 271: 5638-46).

NRPs receptors are transmembrane glycoproteins composed of anextracellular region constituted of two CUB domain (a1 and a2), twoFactor V/VIII (b1 and b2) homology domain and a MAM domain,intracellularly they present a PDZ domain. The structures of domains a2,b1 and b2 of NRP1/2 in the free form (Lee et al., Structure 2003; 11:99-108; Appleton et al., Embo J 2007; 26: 4902-12; Vander Kooi et al.,Proc Natl Acad Sci USA 2007; 104: 6152-7), complexed to tuftsin (VanderKooi et al., Proc Natl Acad Sci USA 2007; 104: 6152-7) and to anantibody (Appleton et al., Embo J 2007; 26: 4902-12) have been reported.

VEGF and Pathological Angiogenesis

The role of VEGF in different pathologies has been reported and blockingthe interaction of VEGF with its receptors has been demonstrated to haveseveral therapeutic applications. Many reviews and patents describe therole and the usage of VEGF inhibitors in pathological angiogenesis anddiscuss their therapeutic applications. All patent applications, patentsand publications cited are hereby incorporated by reference in theirentirety

A diseases which can benefit from a therapy based on the inhibition ofthe interaction between VEGF and its receptors is cancer (D. J. Hicklin& L. M. Ellis J. Clin. Onc. 2005, 23, 1011; N. Ferrara et al., Nat. Med.2003, 9, 669; N. Ferrara & T. Davis-Smyth Endocr. Rev. 1997, 18, 4).VEGF is overexpressed in several type of tumors (lung, thyroid, breast,gastrointestinal, kidney, ovary, uterine cervix, carcinomas,angiosarcomas, germ cell tumors, intracranial). VEGF receptors areoverexpressed in some type of tumors, such as, non-small-cell lungcarcinoma, melanoma, prostate carcinoma, leukemia, mesothelioma, breastcarcinoma (D. J. Hicklin & L. M. Ellis J. Clin. Onc. 2005, 23, 1011),and on the surface on angiogenically active endothelial cells.

VEGF is implicated in intraocular neovascularization which may lead tovitreous hemorrhage, retinal detachment, neovascular glaucoma (N.Ferrara et al., Nat. Med. 2003, 9, 669; N. Ferrara Curr. Opin. Biotech.2000, 11, 617) and in eye disorders such as age related maculardegeneration and diabetic retinopathy (US 2006/0030529).

VEGF is also implicated in the pathology of female reproductive tract,such as ovarian hyperstimulation syndrome and endometriosis.

VEGF has been implicated in psoriasis, rheumatoid arthritis (P. C.Taylor Arthritis Res 2002, 4, S99) and in the development of brainedema.

Diseases caused by a defective angiogenesis can be treated (therapeuticangiogenesis) with agents able to promote the growth of new collateralvessels. The VEGF-induced angiogenesis has several therapeuticapplications. Of course, molecules which bind to VEGF receptors andmimic the biological activity of VEGF are useful for the treatment ofthese diseases.

VEGF has been used for the treatments of ischemic cardiovasculardiseases to stimulate the revascularization in ischemic regions, toincrease coronary blood flow and to prevent restenosis afterangioplasty. (M. Simons & J. A. Ware Nat. Rev. Drug Disc. 2003, 2, 1; N.Ferrara & T. Davis-Smyth Endocr. Rev. 1997, 18, 4).

VEGF and its receptors have been implicated in stroke, spinal cordischemia, ischemic and diabetic neuropathy. VEGF is a therapeutic agentfor the treatment of neuron disorders such as Alzheimer disease,Parkinson's disease, Huntington disease, chronic ischemic brain disease,amyotrophic later sclerosis, amyotrophic later sclerosis-like diseaseand other degenerative neuron, in particular motor neuron, disorders (US2003/0105018; E. Storkebaum & P. Carmeliet J. Clin. Invest. 2004, 113,14).

VEGF has a basic role in bone angiogenesis and endochondral boneformation. These findings suggest that VEGF may be useful to promotebone formation enhancing revascularization. Conditions which can benefitfrom a treatment with VEGF are bone repair in a fractures, vertebralbody or disc injury/destruction, spinal fusion, injured meniscus,avascular necrosis, cranio-facial repair/reconstruction, cartilagedestruction/damage, osteoarthritis, osteosclerosis, osteoporosis,implant fixation, inheritable or acquired bone disorders or diseases(US2004/0033949).

VEGF has been implicated in the process of gastric ulcer (Ma et al.,Proc. Natl. Acd. Sci. USA 2001, 98, 6470) wound healing, diabetic footulcers and diabetic neuropathy.

VEGF has been implicated in neurogenesis (K. Jin et al., Proc. Natl.Acd. Sci. USA 2002, 99, 11946) and for the treatment of pathological andnatural states benefiting from the formation or regeneration of newvessels (US 2005/0075288)

VEGF or molecules able to bind to VEGF receptors can be useful for thediagnosis of pathologies which present a overexpression of VEGFreceptors (Li et al., Annals of Oncology 2003, 14, 1274) and to imagingangiogenic vasculature (Miller et al., J. Natl. Cancer Inst. 2005, 97,172).

Molecular agents for imaging angiogenesis must bind to the VEGFreceptors with high specificity and be detectable at low concentrations.They should be labeled according to the imaging modalities, PET, SPECT,and, to a lesser extent, ultrasound (with microbubble contrast agents)and optical imaging (with fluorescent contrast agents). In addition,even though the sensitivity of MRI is low, molecular imaging ofangiogenesis is possible with oligomerized paramagnetic substanceslinked to an agent, that binds a molecular marker of angiogenesis(Miller et al., J. Natl. Cancer Inst. 2005, 97, 172).

Therefore, there is the need to develop compounds able to modulateVEGF-dependent diseases as therapeutic, diagnostic or imaging agents.

This invention relates to synthetic peptides designed to mimic the VEGFβ-hairpin region 79-92 (or 87-100 of PlGF) able to bind to the VEGFreceptors and to modulate the angiogenesis mediated by the VEGF.

The compounds described in the present invention promote or inhibit theangiogenesis process and have application in the pro- oranti-angiogenesis therapy. Moreover, they are useful as diagnosticagents, for example in the imaging of tumor or endothelial cellsoverexpressing the VEGF receptors. Finally, they have application asbiochemical tools for the study of cellular pathway dependent on theactivation of VEGF receptors.

These compounds have been designed starting from the molecular structureof the complex VEGF-VEGFR1-D2 (Wiesmann, C. et al., Cell 1997, 91, 695;Protein Data Bank code 1FLT). The VEGF (UniProtKB/Swiss-Prot P15692)amino acid sequence 79-92 (QIMRIKPHQGQHIG; one-letter amino acid code;SEQ ID No: 6), comprising strands β5 and β6, adopts a β-hairpinconformation both in the VEGF free and complexed form. Mutagenesisexperiments showed that residues Gln79, Ile83, Lys84 and Pro85 areimportant for VEGFR2 recognition. From the structural analysis of thecomplex VEGF-VEGFR1-D2 the authors identified the VEGF residues (Gln79,Met81, Ile83, Pro85, His86, Gln89, Ile91) situated at less than 4.5 Åfrom the receptor. A corresponding analysis on the PlGF(UniProtKB/Swiss-Prot P49763; sequence 87-100: QLLKIRSGDRPSYV; SEQ IDNo: 7) highlighted the residues Gln87, Leu89, Ile91, Pro97 and Tyr99.

The design strategy consisted in keeping fixed the tridimensionalarrangement of the interacting residues constraining the amino acidsequence 79-92 to adopt a molecular conformation like the natural one.To stabilize the beta-hairpin structure and considering that turnresidue cannot be modified because they are involved in the binding tothe receptor, the authors decided to introduce in the amino acidsequence, where possible, amino acids with high propensity for a betaconformation and stabilize the fold of the two strands using twomolecular tools: hydrophobic cluster and disulfide bond. The firststrategy consisted in introducing, in specific position across the twostrands, hydrophobic amino acids, especially aromatic, in order to forma hydrophobic cluster which keep the two strand folded (Cochran et al.,Proc Natl Acad Sci USA. 2001; 98: 5578-83). The latter strategyconsisted in introducing two cysteine residues in order to form adisulfide bond between the two strands. Polar residue with highpreference for a beta conformation, such as serine and threonine, werealso introduced in order to stabilize the β-hairpin and improve watersolubility. Finally, a lysine residue was appended in order to completestrand pairing and allow peptide derivatization.

SUMMARY OF THE INVENTION

It is therefore the object of the present invention a molecule ofgeneral formula (1) (SEQ ID No 5):X₁Y₁X₂Y₂X₃Y₃X₄Y₄Y₅X₅X₆Y₆X₇Y₇X₈X₉X₁₀  (10)wherein

X1-X10 are any natural or unnatural amino acids and Y1 is Gln; Y2 is Metor Leu;

Y3 is Ile; Y4 is Pro or Ser; Y5 is His or Gly; Y6 is Gln or Pro; Y7 isIle or Tyr or their homolog or ortolog.

Preferably X2, X3, X7 and X8 are amino acids that stabilize thestructure of the molecule.

Still preferably X2, X3, X7 and X8 are selected from the group of: Cys,Trp, Tyr, Ile, Glu, Lys, Phe, Asp.

Yet preferably X4, X5 and X6 are residue present in the natural proteinVEGF or PlGF such as Arg, Lys, Gln, Asp, Gly, Arg.

Preferably X9 and X10 are Ser and Thr, respectively.

Preferably the molecule is selected from the group of: KQIMWIKPHQGQWIYTS(SEQ ID No 1); KQCMWIKPHQGQWTCTS (SEQ ID No 2); KQLLWIRSGDRPWYYTS (SEQID No 3); KQCLWIRSGDRPWYCTS (SEQ ID No 4).

Preferably, the molecule of the invention is for medical use, inparticular for use of a VEGF-dependent pathology.

Preferably the pathology is selected from the group of: cancer, eyedisorders, pathology of female reproductive tract, psoriasis, rheumatoidarthritis, brain edema, diseases caused by defective or excessiveangiogenesis, ischemic cardiovascular diseases, brain disorders, bonepathology, osteoarthiritis, osteosclerosis, esteoporosis.

It is a further object of the invention the use of the molecule of theinvention for the diagnosis of a VEGF-dependent pathology.

It is further object of the invention the use of the molecule of theinvention as an imaging agent. Preferably the molecule is labelled.

Still preferably the label is a contrast or a paramagnetic agent.

Preferably angiogenic vasculature is imaged.

It is further object of the invention a pharmaceutical compositioncomprising the molecule of the invention as described above andappropriate diluents and/or excipients.

In the general formula (1) (SEQ ID No 5):X₁Y₁X₂Y₂X₃Y₃X₄Y₄Y₅X₅X₆Y₆X₇Y₇X₈X₉X₁₀  (1)

X residue are important for the conformational stabilization of themolecule while Y residues are interacting residues.

X1-X10 are any natural or unnatural amino acids, including D-aminoacids: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, Val, α-amino-glycine, α,β-diaminopropionicacid, ornitine, 2,6-diamino-4-hexynoic acid, 4,5-dehydro-lysine,ω-hidroxy-norarginine, α,γ-diaminobutyric acid,α-difluoromethyl-ornitine, 4-amino-piperidine-4carboxylic acid,d-hydroxy-lysine, homoarginine, dehydroalanine, β-cyclopropil-alanine,β-(1-cyclopentenyl)-alanine, β-cyclohexyl-alanine, penicilamine,β-fluoro-alanine, tert-butyl-alanine, β-cyclopentenyl-alanine,β,β-dicyclohexyl-alanine, 4,5-dehydro-leucine, β-chloro-alanine,propargyl-glicine, thioproline, 4-fluoro-proline, allo-isoleucine,azetidine-2-carboxylic acid, 2-allyl-glycine, 3,4 dehydro-proline,α-methyl-proline, pipecolinic acid, α-aminobutyric acid,tert-butyl-glicine, α-methyl-valine, norvaline, cyclohexil-glicine,β-cyano-alanine, allo-threonine, hydroxy-proline,6-diazo-5-oxo-norleucine, β-(2-thiazolyl)-alanine, α-amino-isobutyricacid, β-ureido-alanine, citrulline, homocitrulline,β-(1,2,4-triazol-1-yl)-alanine, homocysteine, pyroglutamic acid,thiocitrulline, S-methyl-thiocitrulline, β-(2-thienyl)-serine,γ-hydroxy-glutamic acid, γ-methylene-glutamic acid, γ-carboxy-glutamicacid, α-aminoadipic acid, 2-aminoheptanedioic acid, α-aminosuberic acid,ω-amino-arginine, α-methyl-histidine, 1-methyl-histidine,β-(2-pyridyl)-alanine, β-(2-Quinolyl)-alanine, ω-methyl arginine,2,5-diiodo-histidine, 3-methyl-histidine, β-(3-pyridyl)-alanine,3-amino, tyrosine, 1-amino-cyclopentanecarboxilic acid,β-(2-thienyl)-alanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,spinacine, 4-amino-phenylalanine, α-methyl-leucine,β-(3-benzothienyl)-alanine, β-(1-naphtyl)-alanine,β-(2-naphthyl)-alanine, β,β-diphenyl-alanine, norleucine, phenilglycine,4-iodo-phenilalanine, 3,4-dichloro-phenylalanine,4-methyl-phenylalanine, octahydrohyndole-2-carboxylic acid,4-bromo-phenylalanine, 3-fluoro-phenilalanine, 4-chloro-phenilalanine,4-phenyl-phenylalanine, α-methyl-phenylalanine, homophenilalanine,4-tert-butyl-phenylalanine, 4-fluoro-phenilalanine,2,3,4,5,6-pentafluoro-phenylalanine, 2-mercapto-histidine,4-azido-phenylalanine, α-methyl-tryptophan, 4-methyl-tryptophan,4-benzoyl-phenylalanine, 4-nitro-phenylalanine, 4-cyano-phenylalanine,3-iodo-tyrosine, 5-methyl-tryptophan,1,2,3,4-tetrahydronorharman-3-carboxylic acid,β-(3,4-diihydroxy-phenyl)-serine, 3,5-dinitro-tyrosine,3-nitro-tyrosine, 7-hydroxy-1,2,3,4-tetrahydroiso-quinoline-3-carboxyacid, 3-hydroxylmethyl-4-isopropylidene-tyrosine,4-carboxy-phenylalanine, 3,5-dibromo-tyrosine, 3,5-diiodo-tyrosine,thyronine, aminohexanoic acid, aminoserine, butylglicine,dihydroxyphenilalanine, homoserine, isonipecotic acid, sarcosine,

Y1 is Gln;

Y2 is Met or Leu;

Y3 is Ile;

Y4 is Pro or Ser;

Y5 is His or Gly;

Y6 is Gln or Pro;

Y7 is Be or Tyr

Preferably positions Y1 to Y7 correspond, but are not limited, to humanVEGF residues Gln79, Met81, Ile83, Pro85, His86, Gln89, Ile91, humanPlGF residues Gln 87, Leu89, Ile91, Ser93, Gly94, Pro97 and Tyr99 or atheir combination. Obviously, the Y residues could also be derived byany combination of residues belonging to a VEGF homolog(VEGF-B/UniProtKB/Swiss-Prot P49765, VEGF-C/UniProtKB/Swiss-Prot P49767,VEGF-D/UniProtKB/Swiss-Prot O43915) or ortholog.

Preferably in positions X2, X3, X7 and X8 are inserted amino acid inorder to conformational stabilize the β-hairpin structure by means ofcovalent bond or non covalent interactions, for example residues X2, X3,X7 and X8 could be any of the following: Cys, Trp, Tyr, Ile, Glu, Lys,Phe, Asp. X4, X5 and X6 preferably are residue present in the naturalprotein VEGF or PlGF such as Arg, Lys, Gln, Asp, Gly, Arg. Preferredamino acid in position X9 and X10 are those with a high preference for abeta conformation (Smith et al., Biochemistry 1994, 33, 5510; Minor L. &Kim P S Nature 1994, 367, 660): Ile, Met, Phe, Ser, Thr, Trp, Tyr, Val,preferably Ser and Thr.

Preferred peptide sequences are:

(SEQ ID No 1) #1 KQIMWIKPHQGQWIYTS (linear) (SEQ ID No 2) #2KQCMWIKPHQGQWTCTS (cyclic: Cys residues make a disulfide bond)(SEQ ID No 3) #3 KQLLWIRSGDRPWYYTS (linear) (SEQ ID No 4) #4 KQCLWIRSGDRPWYCTS (cyclic: Cys residues make a disulfide bond)

The present invention comprises also derivatives of the general molecule(1) or peptides #1-#4 as their cyclic analogs where cyclization couldinterest the backbone, backbone and side chain or side chain-side chain.Other analogs derive by introduction (or replacement) of a covalent bondacross two residues belonging to the two strands. The cyclizationlinkage could be, but are not limited to: lactam bond, carbon-carbonbond, thioether bond, ether bond, an oxime linker, a lanthionine linker,diaryl ether or diarylamine linkage, thiazolidine linkage or as resultof reaction such as click chemistry (Angell Y L, Burgess K. Chem SocRev. 2007; 36: 1674-89), Staudinger ligation (Nilsson et al., Org Lett.2000; 2: 1939-41; Saxon et al., Org Lett. 2000; 2: 2141-3), chemicalligation (Dawson and Kent Annu Rev Biochem. 2000; 69: 923-60), OlefinMetathesis (Schuster et al, Angewandte. Chem. Int. Edn Engl,36:2036-2056 (1997)).

The β-harpin structure could also been stabilized by introducing organicβ-turn mimetic or inducer to chemically connect the peptide N- andC-termini.

The present invention comprises also other derivatives in which one ormore amino acid side chains are protected with a protecting group,peptide N- and C-termini are free, capped with acetyl, amide, ester,thioester, acyl, alkyl, carboxyl, amine, ect. groups, or presentadditions or truncations in the N- and/or C-termini.

It is also within the scope of the invention the modification oroptimization of the described peptides in order to increase theirpotency, pharmacokinetic behavior, stability and/or other biological,physical and chemical properties.

Possible modification, for example, are:

Substitution of the backbone amide bonds with the insertion ofalpha-N-methylamides, thioamides, amines, semicarbazone derivatives,olefin bond; substitution of L-amino acid with a D-amino acid,substitution of alkyl-substituted hydrophobic amino acids with analiphatic side chain from C1-10 carbons including branched, cyclic andstraight chain alkyl, alkenyl or alkynyl substitutions; substitution ofaromatic amino acids; substitution of amino acids containing basicfunctions; substitution of acidic amino acids; substitution of sidechain amide residues, substitution of hydroxyl containing amino acids;

Are also in the scope of the present patent application a retroinversepeptide, a peptoid, multimeric constructs of the described sequences. Inaddition, are also included derivatives of the described sequences whichfunctionalized with a chemical entity such as, but not limited to,glycosyl, PEG, lipid, a metal chelating unit, a fluorescent probe,biotin-derivatives.

Biological and in vivo assays showed that molecules based on the generalformula (1) are able to bind to VEGF receptors and modulate theangiogenic response. The authors found that the compound #3(KQLLWIRSGDRPWYYTS) has a proangiogenic biological activity whereascompounds #1, #2 and #4 showed an antiangiogenic activity. The compoundshave application in diagnosis and treatment of pathologies depending onangiogenesis, especially they can be used in therapy of angiogenesis.

The compounds have applications in diagnosis and treatment ofpathological states depending on excessive angiogenesis, of pathologicaland natural states benefiting from the formation or regeneration of newvessels, of pathologies which present a overxpression of VEGF receptors.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be now described by non limiting examples referringto the following figures:

FIG. 1—HUVE cells (1×10⁴/cm²) were incubated in starvation medium(EBM-2, 0.1% heparin, 0.1% BSA) with A) peptide #3 (25-100 ng/ml) orscrambled peptide pep-RND (100 ng/ml); B) peptide #4 or C) peptide #2 at37° C. in 5% CO2 atmosphere. VEGF165 was used as positive control (seemethods). After 8 hours, caspase-3 activity was determined. Data arereported as percent of cell rescue from caspase-3 activation withrespect to control.

FIG. 2—HUVE cells were incubated in starvation medium (EBM-2, 0.1%heparin, 0.1% BSA) with VEGF (25 ng/ml) or A) peptide #3 (25-100 ng/ml),scrambled Pep-RND (100 ng/ml) peptides for 15 min and 30 min; B) in theabsence or presence of peptide #4 (100 ng/ml) for 30 min. The experimentwas performed at 37° C. in 5% CO2 atmosphere. Proteins were analyzed forphospho ERK1/2 content by immunoblotting. An antibody recognizing GAPDHwas used to monitor equal loading conditions.

FIG. 3—A) HUVEC cells were incubated in starvation medium (EBM-2, 0.1%heparin, 0.1% BSA) alone or in presence of VEGF165 (25 ng/ml) or withindicated concentrations of Pep #3 or Pep RND (100 ng/ml), for 24, 48and 72 hours at 37° C. in 5% CO2 atmosphere. Cells were then incubatedwith CyQUANT® NF reagent for 1 hour at 37° C. and fluorescenceintensities of quadruplicate samples were measured with a fluorescencemicroplate reader using excitation at 485 nm and fluorescence detectionat 530 nm. In graph values are calculated as % of proliferating cellscompared to controls (untreated cells) at 24, 48 and 72 hours. B) ERK1/2phosphorylation and phospho-RB was evaluated at 24 hr after stimulationwith Pep #3 (10-25-50 and 100 ng/ml), VEGF165 (25 ng/ml) and Pep RND(100 ng/ml). Anti-ERK1/2 and anti-GAPDH antibodies were used as loadingcontrol

FIG. 4—Angioreactors were prepared and implanted as described in themethod section. After 21 days, angioreactors were recovered andphotographed using a digital camera (A). In VEGF- and peptide#3-containing angioreactors, new vessel formation was observed. Panel B)Blood vessel development was quantified by fluorescence microscope bydetermining the number of FITC-lectin-positive cells. 20 fields forsample were analyzed; results are expressed in number of FITC-lectinpositive cells/field.

DETAILED DESCRIPTION OF THE INVENTION

Material and Methods

Peptide Synthesis

Peptides were synthesized on solid phase using Wang resin (Novabiochem)with standard Fmoc (N-(9-Fluorenyl)methoxycarbonyl) chemistry. TheN-terminal lysine was protected with the methyltrytil group to allowselective deprotection and peptide labeling. Cleavage from the resinwere achieved by treatment with trifluoracetic acid, triisopropylsilane, water, etanedithiol (93; 2, 2.5; 2.5) at room temperature for 3hours. Purity and identity of the peptides were assessed by HPLC andMALDI-ToF mass spectrometry. The VEGF used as positive control was thecommercial available VEGF₁₆₅ (UniProtKB/Swiss-Prot P15692-4)

Cell Culture

HUVEC (Human Umbilical Vein Endothelial Cells) were purchased fromPromocell (Heidelberg, Germany). All experiments were performed on lowpassage cell cultures. Cells were grown in EGM-2 (Endothelial Growthmedium) (EBM-2, FBS 2%, VEGF, R3-IGF-1, hEGF, hFGF, hydrocortisone,ascorbic acid, heparin and GA-1000) (Clonetics, Cambrex Bio ScienceWalkersville, Inc., Walkersville Md. USA) at 37° C. and in 5% CO2.

Casapase 3 Fluorimetric Assay

Determination of caspase-3 activity was performed by a fluorometricassay based on the proteolytic cleavage of the AMC-derived substrateN-acetil-DEVD-AMC, which yields a fluorescent product.

HUVEC cells were plated in 6-well dishes at 1×10⁵ cells/cm². On the nextday, cells were treated, in starvation medium (EBM-2, heparin 0.1%, BSA0.1%), with Pep #3 (25-100 ng/ml) or scrambled Pep RND (100 ng/ml)peptides for 8 h at 37° C. VEGF165 (R & D Systems, Minneapolis, Minn.,USA), 25 ng/ml, was used as positive control.

After 8 h the cells were processed with 150 μl of Caspase-3 reactionbuffer (HEPES pH 7.5 50 mM, EDTA 0.1 mM, NP-40 0.1%, CHAPS 0.1%, DTT 1mM) and cell proteins collected after centrifugation at 13000 rpm for 15min at 4° C. Protein concentrations were determined by Bradford method(Bio-Rad, Hercules, Calif.) and 20 μg of lysates were incubated in96-well plates with 20 μM N-acetil-DEVD-AMC at 37° C. for 3 h.

Samples were analyzed using a microplate reader (L55 LuminescenceSpectometer Perkin Elmer Instruments) (excitation: 360 nm, emission: 440nm).

Western Blot

Cells were plated in 12-well dishes at 1×10⁵ cells/cm². On the next day,cells were treated, in starvation medium (EBM-2, heparin 0.1%, BSA 0.1%)with Pep #3 (25-100 ng/ml) or Pep RND (100 ng/ml) peptides for 15 minand 30 min at 37° C. VEGF165 (R & D Systems, Minneapolis, Minn., USA) 25ng/ml was used as positive control. After treatment, whole cell lysateswere obtained by using RIPA lysis buffer (Tris 50 mM pH 7.5, NaCl 150mM, NP-40 1%, EGTA 1 mM, SDS 0.05%) supplemented with Complete ProteaseInhibitors and Phosphate Inhibitors (Pierce Biotechnology, Rockford,Ill.). Cell proteins were then collected after centrifugation andprotein content was determined by Bradford method (Bio-Rad, Hercules,Calif.). Proteins were separated by electrophoresis onSDS-polyacrylamide gels and transferred to nitrocellulose membrane(Millipore Corp., Bedford, Mass.) by semi-dry electroblotting(Transblot; Bio-Rad Laboratories, Hercules, Calif.).

The serine-tyrosine-phosphorilated ERK1/2 (polyclonal antibody, CellSignaling Technology, Inc. Danvers, Mass., USA) and αGAPDH (monoclonalantibody 6C5, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) werevisualized by specific antibodies, anti-rabbit and anti-mousehorseradish peroxidase-conjugated secondary antibody (JacksonImmunoresearch Laboratories, Inc, Baltimor Pike; Pa., USA) standardchemiluminescence (Pierce Biotechnology, Rockford, Ill.).

Cell Proliferation Assay

HUVEC cells were plated at density of 1200 cells/well in 96-wellpoly-D-lysine-coated microplates (Becton Dickinson, Franklin Lakes, N.J.USA). After 24 hours incubation in starvation medium (EBM-2, heparin0.1%, BSA 0.1%), cells were treated with VEGF165 25 ng/ml (R & DSystems, Minneapolis, Minn., USA), with Pep #3 (10-25-50-100 ng/ml) orPep RND (100 ng/ml). Cell proliferation was determined by using CyQUANT®NF Cell Proliferation Assay Kit (Molecular Probes, Invitrogen S.R.L.,Milano, Italy) at 24, 48 and 72 hour after treatment. CyQUANT® NF assayis based on cellular DNA content measurement via fluorescent dyebinding. Because cellular DNA content is highly regulated, it is closelyproportional to cell number. Briefly, medium was removed and cellsincubated with CyQUANT NF reagent for 1 hour at 37° C. according to themanufacturer's instructions. Plates were then analyzed by using amicroplate reader (L55 Luminescence Spectometer Perkin ElmerInstruments) (excitation: 485 nm, emission: 520 nm).

Animals

Female CD1 mice (20-25 g) were supplied by Charles-River (Italy) andkept in temperature- and humidity-controlled rooms (22° C., 50%) withlights on from 07:00 to 19:00 h, water and food available ad libitum.All procedures were carried out in accordance with the Italian law(Legislative Decree no. 116, 27 Jan. 1992), which acknowledges theEuropean Directive 86/609/EEC, and were fully compliant withGlaxoSmithKline policy on the care and use of laboratory animal andcodes of practice. Furthermore, all efforts were made to minimize thenumber of animals used and their suffering.

Directed in Vivo Angiogenesis Angioreactor (DIVAA)

Sterile silicone cylinders closed at one end, called angioreactors(Cultrex, Trevigen, Inc. Helgerman Conn., Gaithersburg), were filledwith 20 μL of basement membrane extract (BME) premixed with or withoutangiogenesis factors (VEGF, FGF) to obtain positive and negativecontrols respectively. Furthermore HPLW (100 ng/ml) or scrambled HW-RND(100 ng/ml) peptides were added to angioreactors. These were incubatedat 37° C. for 1 hour to allow gel formation, before subcutaneousimplantation into the dorsal flank of CD1. Animals were anaesthetizedbefore implantation with 100 mg/ml Ketamine HCL and 20 mg/ml Xylazineinjected subcutaneously. Incision were made on the dorsal flank (leftand right) of mouse, approximately 1 cm above the hip-socket, and wereclosed with skin staple.

Vessel formation evaluation was performed after 21 days. Matrigel wasremoved from angioreactors and digested in 300 μl of CellSperse solutionfor 1 hour at 37° C. After digestion, the incubation mix was cleared bycentrifugation at 800 rpm. Cell pellets were resuspended in 500 μl ofDMEM 10% FBS and plated on coverslips in 24-wells plates for 16 hour at37° C. in 5% CO2. Cells were fixed with a 3.7% formaldehyde solution for30 min at room temperature and quenched by incubation with 0.1 mMglycine for 5 min. Subsequently, cells were incubated with FITC-Lectinreagent (available in the kit) and then observed by fluorescencemicroscope (ZEISS, German).

Results

Peptides Biological Effects on Caspase 3 Activity

To investigate the biological activity of the designed peptides theauthors analyzed the activation of caspase 3 in human primaryendothelial (HUVEC) cells serum-deprived by means of a caspase-3fluorimetric assay. It is well known that VEGF act as survival factorfor ECs. When serum starved caspase 3 activity increases, therefore, theaddition of VEGF partially rescued, as expected (Yilmaz A, et al.,Biochem Biophys Res Commun. 2003, 306: 730-6), HUVEC cells fromapoptosis. Experiments were performed on serum starved HUVEC treatedwith designed peptides in presence or absence of VEGF in order tohighlight an antagonist or agonist (VEGF-like) activity respectively.The biological activity of the analyzed compounds is reported aspercentage of VEGF rescue (FIG. 1). The decrease of the caspase-3activity following VEGF incubation is set to 100% rescue. Caspase-3activity, measured after 8 hour, was significantly decreased (andconsequently the rescue was significantly increased) by Peptide #3 atfinal concentrations of 25 ng/ml and 100 ng/ml, in respect to untreatedcells and scrambled (Pep-RND)-treated cells.

The effect of VEGF was partially abolished when peptide #2 and #4 wereadded to the cultures (FIGS. 1B and 1C).

Biological Effects of Peptides #3 and #4 on ERK 1/2 and AKT Activation

VEGF-modulated angiogenesis is largely ERK1/2-dependent, leading to DNAsynthesis and cell proliferation. VEGF binding to HUVEC cells was shownto induce the activation of ERK kinase. The authors thereforeinvestigated whether peptide #3 could also induce ERK and AKT activationlike VEGF. HUVEC cells, treated with peptide #3 (25-100 ng/ml) in serumdeprivation conditions for 15 and 30 min, displayed ERK1/2 and AKTactivation, as shown in western blot analyses by using an anti-phosphoserine-tyrosine ERK1/2 and AKT antibodies (FIG. 2A). Scrambled Pep-RNDhad no effect on ERK1/2 and AKT activation, proving that it is unable toactivate intracellular signaling (FIG. 2A).

Peptide #4, instead, at 100 ng/mL does not show any biological effect onuntreated HUVE cells but dramatically reduces ERK 1/2 activation of VEGFstimulated HUVE cells, confirming its biological inhibitory activity(FIG. 2B).

Effect of Peptide #3 on Cell Proliferation

To evaluate whether peptide #3 induces cellular proliferation, a cellproliferation assay was performed on HUVEC cells treated, in serumdeprivation conditions, with pep #3 (10-25-50-100 ng/ml) or Pep RND (100ng/ml). After 24, 48 and 72 hours of treatment cell proliferation wasmeasured by CyQUANT® NF Cell Proliferation Assay Kit. Results obtainedfrom this experiment demonstrated that peptide #3 is able to induce, ina dose dependent manner, cell proliferation in a manner similar toVEGF165 (25 ng/ml) used as positive control. This effect is evident at24, 48 and 72 hours. Considerable effects were also observed with pep #3treatment at concentrations of 25 and 50 ng/ml at 48 and 72 hours. Thescrambled peptide, pep RND, was ineffective at the concentration of 100ng/ml. (FIG. 3A).

Moreover, western blot analysis, from whole cells proteins obtain after24 hours of treatment, confirmed ERK1/2 activation in cells treated withpeptide #3 and VEGF165. As a marker of cell proliferation, the RBphosphorylation status was also checked in the same experimentalconditions. RB protein, in fact, is able to regulate proliferation bycontrolling cell cycle progression through the restriction point withinthe G1 and S phases. Pep #3 and VEGF165 but not Pep RND, enhanced RBphosphorylation, thus indicating cell cycle progression from G1 to Sphase (FIG. 3B).

Peptide #3 Pro-Angiogenic Activity In Vivo

Pro-angiogenic peptide #3 activity was also assayed in an in vivo testusing DIVAA (Directed in Vivo Angiogenesis Angioreactor). DIVAA test isperformed by implanting angioreactors into the dorsal flank of mice;this allows a quantitative assessment of blood vessel development. InFIG. 3A are shown pictures from angioreactors containing PBS (negativecontrol), VEGF (positive control), peptide #3 (100 ng/ml) or scrambledPep-RND peptide (100 ng/ml), removed after 21 days from subcutaneousimplantation in mice. Blood vessels development was observed in VEGF-and peptide #3-containing angioreactors. Indeed, as shown in FIG. 3B,the induction of new vessel formation by peptide #3 was 3.8 fold greaterwith respect to negative controls (peptide #3 vs Pep-RND: 3.83±0.67,p=0.000835; peptide #3 vs PBS:2.4±0.42, p=0.001477).

The invention claimed is:
 1. A synthetic peptide comprising the aminoacid sequence selected from the group consisting of: KQCMWIKPHQGQWTCTS[SEQ ID No 2](cyclic: Cys residues make a disulfide bond) andKQLLWIRSGDRPWYYTS [SEQ ID No 3].
 2. A pharmaceutical compositioncompromising the peptide of claim 1 and appropriate diluents and/orexcipients.